LOCUS       sequence-238200-        7336 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression in BL21(DE3) PlysS of mutant T4 DNA polymerase Y320A for 
            easy cloning and mutagenesis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7336)
  AUTHORS   Qi R, Otting G
  TITLE     Mutant T4 DNA polymerase for easy cloning and mutagenesis.
  JOURNAL   PLoS One. 2019 Jan 23;14(1):e0211065. doi: 
            10.1371/journal.pone.0211065. eCollection 2019.
  PUBMED    30673756
REFERENCE   2  (bases 1 to 7336)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2019-01-23"; volume: "14(1):e0211065. doi"; pages: " 
            10.1371/journal.pone.0211065. eCollection 2019"
COMMENT     Sequence Label: pETMCSI-T4P_Y320A
FEATURES             Location/Qualifiers
     source          1..7336
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..70
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     154..173
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        154..172
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             204..226
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(3015..3033)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3029..3076
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3442..3447,3448..3464,3465..3470))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3501..3519)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3587..3691
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3692..3760,3761..4552)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3910..3929)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4723..5311
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5212..5231
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5465..5482
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5497..5636
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5562..5581)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5722..5744
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5738..5929)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
       61 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
      121 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata gggagaccac
      181 aacggtttcc ctctagaaat aattttgttt aactttaaga aggagatata catatgaaag
      241 aattttatat cagcatcgaa accgttggca ataatatcgt tgagcgctac atcgatgaaa
      301 acggcaaaga acgtactcgc gaggtggagt atctgccaac catgtttcgt cactgcaaag
      361 aagagtctaa atataaagac atttatggca agaattgtgc cccacagaaa tttccgtcta
      421 tgaaggatgc gcgtgactgg atgaagcgga tggaggatat cgggctggag gcgttaggca
      481 tgaacgattt taaattagcc tacatttcgg atacctatgg ctccgagatc gtatatgatc
      541 gtaaatttgt tcgcgtagcg aactgtgaca tcgaggtgac gggggataaa ttcccggacc
      601 caatgaaagc agaatatgaa atcgacgcta tcactcacta tgacagtatc gatgaccgct
      661 tctatgtctt cgatttactg aatagcatgt acggttcagt ttccaaatgg gatgcaaaac
      721 tggctgcgaa gcttgattgc gaaggaggtg atgaagttcc gcaggaaatt ctggatcggg
      781 tcatttatat gccattcgat aacgaacgtg acatgctcat ggagtatatc aacctctggg
      841 agcaaaaacg ccccgcgatc tttacaggtt ggaacatcga gggtttcgat gtgccgtaca
      901 ttatgaaccg tgtcaaaatg atccttgggg aacgttctat gaaacgtttc agtccgatcg
      961 ggcgcgtgaa atccaagctt attcaaaata tgtatggaag caaagaaatt tacagcattg
     1021 atggggtttc aatcctggat tatttggacc tttataaaaa attcgctttt actaacctgc
     1081 cgtcattttc gctggaaagt gtagcacagc acgaaacaaa aaaaggtaag cttccgtatg
     1141 atgggccaat caataaactt cgtgaaacga accatcagcg ctatatttcc gcaaacatta
     1201 tcgacgtaga aagcgtgcag gcaatcgata aaattcgcgg ttttattgat ctggtactga
     1261 gcatgtcgta ttatgcaaaa atgccattta gtggtgtaat gagcccgatc aaaacctggg
     1321 atgcgattat cttcaatagc ctgaaaggtg agcataaagt tattccgcag cagggctctc
     1381 acgtgaaaca aagctttccg ggcgcgtttg tgtttgaacc gaaaccaatc gctcgtcgct
     1441 acattatgag ctttgactta acatctctct acccgtccat tattcggcaa gtcaatatca
     1501 gtccggaaac gattcggggc caatttaaag ttcacccgat ccatgagtat attgcaggca
     1561 ccgcgccaaa accgtccgac gaatacagct gctcgccaaa cggttggatg tatgacaaac
     1621 atcaggaagg aattatccca aaagagatcg ctaaagtctt ctttcaacgc aaagattgga
     1681 aaaaaaaaat gtttgcggag gaaatgaacg cggaagcgat taagaaaatt atcatgaaag
     1741 gtgctgggag ctgtagcacg aaaccggagg tggagcgtta tgtgaaattt agcgacgatt
     1801 ttctgaatga actcagcaat tatacggagt ctgttctgaa ctctctgatt gaagaatgtg
     1861 aaaaggcggc gaccctggcg aatacaaatc agttaaatcg gaaaattctt attaatagtc
     1921 tgtatggtgc gctcgggaac atccattttc gttattacga cctgcggaat gccaccgcaa
     1981 ttaccatttt tggccaggtc ggaatccagt ggattgcccg taaaattaat gagtatctca
     2041 acaaggtctg tggcaccaac gacgaagatt tcatcgctgc gggtgataca gatagcgttt
     2101 atgtctgtgt cgacaaggtt attgaaaaag ttgggctgga tcggttcaaa gaacagaacg
     2161 atctcgtaga atttatgaat cagttcggca aaaaaaagat ggagcctatg attgacgtcg
     2221 cttaccgcga gctgtgtgat tacatgaaca atcgcgaaca ccttatgcac atggatcgcg
     2281 aggcaatttc atgtccgcct ctgggctcga aaggtgtagg gggtttctgg aaagcaaaaa
     2341 aacgttatgc gctcaatgtt tacgatatgg aagataaacg cttcgcggag ccgcacctca
     2401 aaatcatggg aatggaaaca cagcagagct ctaccccgaa agctgttcaa gaagccctgg
     2461 aggaaagtat tcgtcgtatt ctgcaagaag gtgaggagag cgtgcaagaa tattacaaga
     2521 actttgaaaa ggagtatcgt cagctggatt acaaagtaat tgcggaggtt aaaactgcaa
     2581 atgacattgc aaaatatgat gataagggtt ggcccgggtt taaatgtcct ttccacattc
     2641 gtggcgtcct gacgtatcgt cgcgccgtca gcgggctggg tgtcgcgccg atcttggacg
     2701 gcaacaaagt tatggtcctc cctctgcgtg aagggaaccc ttttggtgat aaatgtattg
     2761 cgtggccttc gggcaccgaa ttaccgaaag aaatccgttc cgatgtgctt tcgtggatcg
     2821 atcatagcac tttgttccag aaatcttttg ttaaacctct ggcgggcatg tgcgaaagcg
     2881 cgggtatgga ttacgaagaa aaagcgtcac tggactttct gtttggttga attcgagctc
     2941 ccgggtacca tggcatgcat cgatagatcc ggctgctaac aaagcccgaa aggaagctga
     3001 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     3061 cttgaggggt tttttgctga aaggaggaac tatatccgga tatccacagg acgggtgtgg
     3121 tcgccatgat cgcgtagtcg atagtggctc caagtagcga agcgagcagg actgggcggc
     3181 ggccaaagcg gtcggacagt gctccgagaa cgggtgcgca tagaaattgc atcaacgcat
     3241 atagcgctag cagcacgcca tagtgactgg cgatgctgtc ggaatggacg atatcccgca
     3301 agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc
     3361 cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat
     3421 ttaactgtga taaactaccg cattaaagct tatcgatgat aagctgtcaa acatgagaat
     3481 taattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     3541 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3601 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3661 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3721 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3781 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3841 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3901 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     3961 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4021 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4081 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4141 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4201 cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
     4261 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4321 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4381 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4441 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4501 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4561 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4621 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4681 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4741 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4801 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4861 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4921 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4981 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5041 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5101 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5161 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5221 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5281 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5341 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5401 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5461 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     5521 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     5581 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     5641 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     5701 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     5761 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     5821 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     5881 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     5941 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     6001 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     6061 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     6121 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     6181 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     6241 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     6301 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     6361 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
     6421 tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg
     6481 tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc
     6541 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     6601 acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt
     6661 ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt
     6721 taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg
     6781 cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat
     6841 aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc
     6901 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     6961 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     7021 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     7081 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     7141 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gacgctctcc
     7201 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     7261 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     7321 tgccaccata cccacg
//
